Differential Esterase Expression in Leaves of Manihot esculenta Crantz Infected with Xanthomonas axonopodis pv. manihotis

The polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases in plants and to show a differential expression of esterases as markers of pathogenesis in cassava plants (Manihot esculenta Crantz). The characterization of alpha- and beta-esterases from leaves of M. esculenta by the PAGE system was possible using an extraction solution containing two phenol-complexing agents (PVP-40 and sodium metabisulfite), three antioxidant agents (EDTA, beta-mercaptoethanol, and DTT), and one quinone reducer (ascorbic acid). Fourteen esterase isozymes were detected in young unexpanded leaves of M. esculenta cultivars. The inhibition pattern of alpha- and beta-esterases of M. esculenta showed that Est-9 is an arylesterase, and in the unexpanded leaves of the M. esculenta plants infected with Xanthomonas axonopodis pv. manihotis, the Est-7 beta-esterase showed the characteristic staining of an alpha/beta-esterase. This diffrential expression of Est-7 isozyme in young unexpanded leaves of cassava plants can be used as a marker of pathogenesis after infection with X. axonopodis pv. manihotis.     

Tissue-specific esterases in the xiphophorine fish Platypoecilus maculatus,Xiphophorus helleri,and their hybrid

Tissue-specific esterases of the xiphophorine fishes Platypoecilus maculatus (platyfish), Xiphophorus helleri (swordtail), and their F₁ hybrid have been analyzed using disc electrophoresis. Seven esterase zones (resolved into a maximum of nine bands) exist in these fishes, and these have been classified by employing specific inhibitors. Five of the seven zones, EST-1, EST-2, EST-5, EST-6, and EST-7, appeared to be carboxylesterases; while the two remaining zones, EST-3 and EST-4, were classified as cholinesterases. In the liver of the platyfish, all seven esterase zones were detected, while the liver of the swordtail exhibited only five esterase zones. EST-1 and EST-3 were lacking in the liver tissue of the swordtail. All seven esterase loci were expressed in the liver tissue of the F₁ hybrid. The reciprocal crosses gave the same results. In the fin, skin, skeletal muscle, and eye tissues from all three genotypes, three major esterase zones, EST-2, EST-5, and EST-7, were detected. In addition, EST-1 was frequently detected in all these tissues of the platyfish and the F₁, but was lacking in the swordtail. Serum from three genotypes showed one prominent esterase zone, EST-5; however, trace activity of EST-2 and EST-7 zones could also be detected. It seems that in all tissues of the F₁ hybrid there is expression of all the esterase genes from the platyfish. The results of the present study are discussed in comparison to those from other studies on teleost esterases.This research was supported by grants from the Sonderforschungsbereich 103 Zellenergetik und Zelldifferenzierung (Marburg). M. R. A. is a Richard-Merton Guest Professor supported by the Deutsche Forschungsgemeinschaft.     

Differential gene expression for isozymes in somatic mutants of Vitis vinifera L. (Vitaceae)

Isozyme electrophoresis was used as a method to provide a measure of relationship among Italia, Rubi, Benitaka, and Brasil cv of Vitis vinifera traditionally grown in Marialva, a town in the northwestern region of the state of Paraná, southern Brazil. No allelic variation was observed for esterase (EST), malate dehydrogenase (MDH), peroxidase (POD), glutamate dehydrogenase (GTDH), alkaline phosphatase (AKP), acid phosphatase (ACP), and aspartate amino transferase (AAT). Tissue specific and variation in staining intensity of EST, MDH, POD, and GTDH isozymes indicate differential gene expression in colour grape varieties. Regulatory genes may be operative in determining the number of molecules of enzymes in a cell and determining the berry skin polymorphism in four cultivars. Change frequency for berry skin colour suggest the occurrence of somatic crossing-over in naturally cultivated plants and a periclinal chimerism in Brasil cv. The four grape colour cultivars seem to be clones of the same cultivar.     

Isozyme gene markers in Vicia faba L.

Summary This study was conducted to assess the genetic basis of the variability observed for the glutamate oxaloacetate transaminase (GOT), Superoxide dismutase (SOD), esterase (EST), and malate dehydrogenase (MDH) isozyme systems in different open-pollinated Vicia faba varieties. Individual plants showing contrasting zymogram patterns were simultaneously selfed and cross-combined. Crossing was unsuccessful in producing progeny, and only selfed progenies were suitable for genetical analysis of isozyme variability. Three zones of GOT activity were made visible. The isozyme of GOT-2 and GOT-3 zones were dimeric and under the control of three alleles at the Got-2 locus and two alleles at the Got-3 locus, respectively. The isozymes of the GOT-1 zone did not show any variability. Three zones of SOD isozyme activity were made visible. The isozymes occurring in the SOD-1 (chloroplastic isozyme form) and SOD-2 (cytosol isozyme form) zones were dimeric and under the control of two alleles at the Sod-1 and Sod-2 loci. The isozyme visualized in the SOD-3 zone (mitochondrial isozyme form) were tetrameric and under the control of two alleles at the Sod-3 locus. Apparently the isozymes made visible in the most anodal esterase zones EST-1, EST-2, and EST-3 were monomeric, and the occurrence of two alleles at each of two different loci explained the variability observed in the EST-2 and EST-3 zones. For MDH, only two five-banded zymogram pattern types were found, and every selfed progeny showed only one of the two zymogram type, indicating that each individual possessed fixed alleles at the loci controlling MDH isozyme. Got-2, Got-3, Sod-1, Sod-2, and Sod-3 appear to be five new isozyme gene markers that can be useful in Vicia faba breeding for linkage study, varietal fingerprinting, outcrossing rate estimate, and indirect selection for quantitative characters.     

Genetics of esterase isoenzymes in Malus

Summary Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

葡萄初级核心种质资源MybA基因型分析

探究MybA类基因在不同类型葡萄品种中的分布,可为葡萄品种鉴定,以及有色葡萄育种的亲本选择提供依据。本研究以欧亚种、欧美杂种、法美杂种、山欧杂种以及美洲种在内的118个葡萄初级核心种质为材料,对其MybA基因型进行分析。结果表明:欧亚种及其杂种普遍具有VvmybA1基因的等位基因VvmybA1a,仅10个欧亚种及其杂种品种中没有检测到VvmybA1a基因;欧亚种、欧美杂种以及法美杂种中普遍同时具有VvmybA1、VvmybA2和VvmybA3基因,仅少数品种未检测到VvmybA2或VvmybA3基因;山欧杂种中北玫、公酿1号和熊岳白葡萄同时具有VvmybA1、VvmybA2和VvmybA3基因,北醇和北红中仅检测到VvmybA1和VvmybA3基因;仅在具有美洲种血缘的葡萄品种中检测到VlmybA2基因,而5个认为是美洲种的品种未检测到VlmybA2基因,且检测到了欧亚种特有的VvmybA1a等位基因,推测它们为含美洲种血缘较多的欧美杂种,而非纯美洲种。     

Rat liver microsomal progesterone metabolism: evidence for differential troleandomycin and pregnenolone 16 alpha-carbonitrile inductive effects in the cytochrome P-450 III family

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Linkage Relationships and Chromosome Assignment of Four Esterase Loci in the Mosquito ANOPHELES ALBIMANUS

Although anopheline mosquitoes are important vectors of malaria, their genetic makeup has not yet been extensively investigated. The present studies concentrate on the genetic basis of esterases in Anopheles albinomanus. Nine zones of esterase activity activity have been resolved by gel electrophoresis. Four of these esterases: EST-2, EST-4, EST-6, and EST-8 are present throughout all developmental stages and also posess allelic variation. Mass matings were carried out with homozygous males and females heterozygous for two or more loci. The analyses of the progeny from single egg batches revealed that the four esterase systems mentioned above are encoded in separate loci with codominant allels. Analyses of two-point and three-point crosses have indicated the following linkage relationships: Est-8--12%--Est-4--22%--Est-2--9%--Est-6. The assignment of this linkage group to chromosome 3 has been accomplished by the use of a Y-2 chromosome translocation.     

Diversification within grapevine cultivars goes through chimeric states.

Vitis vinifera 'Pinot' clones were analysed at 50 microsatellite loci to assess intravarietal genetic diversity. When analysing leaf tissue DNAs, polymorphism mainly resulted from the appearance of a third allele when two were expected for heterozygous loci in a diploid species. The sequencing of the three microsatellite alleles at two loci has confirmed their simultaneous presence in the leaf tissues. A hypothesis explaining the triallelic profiles at a locus is the presence of a periclinal chimera meristem structure, in which genetically different cell layers coexist. The periclinal chimeric state of two Vitis vinifera 'Pinot gris' clones was confirmed by splitting and analysing the genotypes resulting from L1 and L2 cell layers in progeny derived from self-fertilization, in root tissues, and in plants regenerated from somatic embryogenesis. Prevalence of chimerism in polymorphic clones observed in a collection of 145 accessions belonging to 'Pinot gris', 'Pinot noir', Pinot blanc', 'Pinot meunier', and 'Pinot moure' cultivars was demonstrated. The accumulation of somatic mutations and cell layer rearrangements allowed us to deduce the relationships between the various genotypes and to open a way for understanding the diversification process and the phylogeny in the 'Pinot' group.     

Synthesis of specifically deuterium-labelled pregnanolone and pregnanediol sulphates for metabolic studies in humans.

A synthesis is reported of 3beta-hydroxy-5alpha-pregnan-20-one sulphate and the disulphate and 3-monosulphate of 5alpha-pregnane-3beta,20alpha-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3beta-hydroxy-5alpha-25R-spirostan-12-one (hemcogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 3beta-hydroxy-5alpha-[11,11-2H2]pregn-16-en-20-one (VII). Further deuterium atoms were introduced at the 3alpha and 20beta positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3beta-hydroxy-5alpha-[3alpha,11,11-2H3]pregnan-20-one (X; isotopic purity 87.2%) and 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly form the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol 20-acetate (XVII).     

Uniformity of Plants Regenerated by Direct Somatic Embryogenesis from Zygotic Embryos of Trifolium repens

A total of 55 regenerants from direct somatic embryogenesison three zygotic embryos of Trifolium repens have been examinedfor uniformity with respect to morphological markers, chromosomenumber, breeding behaviour and banding patterns produced bySDS-polyacrylamide gel electrophoresis of total leaf proteinsand isoelectric focussing of leaf peroxidase, esterase and leucineaminopeptidase isozymes. Differences were observed between clones,but no differences were detected among primary and secondaryregenerants from the same zygotic embryo. The data support theconcept that direct somatic embryogenesis on immature zygoticembryos is a conservative, clonal regeneration process. Trifolium repens, clover, somatic embryogenesis, somaclonal variation, embryo culture, isozymes     

Histochemical localization of some hydroxysteroid dehydrogenases in the mouse epididymis.

The mouse epididymis was studied to localize histochemically a number of hydroxysteroid dehydrogenases in the various zones. The epithelium of the posterior half of the initial segment (head) and the anterior half of the middle segment (body) shows a strong reaction for delta5-3beta-, 3alpha,5alpha-, 3alpha,5beta-, 11beta, 16alpha-, 17beta, 20alpha-hydroxysteroid dehydrogenases. This activity attenuates posteriorly. Only the 11beta-hydroxysteroid dehydrogenase is present throughout the length of the epididymis. The luminal contents of the middle segment also show the histochemical utilization of a number of steroids.     

Characterization of Esterases in a Brazilian Population of Zaprionus Indianus (Diptera: Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using α- and β-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as β-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as α-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.     

Distribution and probable physiological role of esterases in reproductive,digestive, and fat-body tissues of the adult cotton boll weevil,Anthonomus grandis Boh

Polyacrylamide gel electrophoresis was used to examine gut, Malpighian tube, fat-body, testes, and ovariole tissues of the adult cotton boll weevil, Anthonomus grandis Boh. Esterases for which the inheritance has been reported previously by Terranova using whole-body homogenates were detected in dissected tissues and the probable physiological function of each allozyme is suggested. EST-1 occurs most frequently in ovarioles and female fat bodies. EST-2 is most often found in fat bodies and may be important in lipid turnover. No sex difference was observed. EST-3^S is found in fat bodies and reproductive tissue, while EST-3^F is always located in gut tissues, indicating that EST-3 is not controlled by a single autosomal locus with two codominant alleles as previously reported. EST-4, the most abundant esterase, can be detected in gut tissue at any age and is probably involved in digestion. EST-5 contains four allozymes which appear most frequently in testes and may be important during reproduction.     

Pregnene derivatives from Solenostemma argel leaves

Two new pregnene derivatives 14beta-15alpha-dihydroxy-delta4pregnene-3,20 dione and 3beta-14beta,15alpha-16alpha hydroxy-20-oxo-delta5pregnene-tetra-ol, in addition to alpha- and beta-amyrin and beta-sitosterol, were isolated from Solenostemma argel leaves. The structures were established by extensive spectral analysis as well as comparison with reference materials.     

Epoxidation of androsta-5,16-dien-3 beta-ol by hepatic microsomal lipid peroxidation

Male rat liver microsomes oxidized androsta-5,16-dien-3 beta-ol (delta 16-ANDO) to delta 16-ANDO-5,6 alpha-, -5,6 beta-, -16,17 alpha-, and -16,17 beta-epoxides and delta 16-ANDO-5 alpha,6 beta-, -16 alpha,17 beta-, and -16 beta,17 alpha-glycols in the presence of an NADPH-generating system and the microsomal lipid peroxidation accelerator, Fe2+-ADP. The hepatic microsomes hydrolyzed all the delta 16-ANDO epoxides to the glycols. delta 16-ANDO-5 alpha,6 beta-glycol was the sole metabolite from both 5,6 alpha- and 5,6 beta-epoxides. Microsomal epoxide hydrolase also hydrolyzed delta 16-ANDO-16,17 alpha-epoxide specifically to the 16 beta,17 alpha-glycol and the isomeric 16,17 beta-epoxide to the 16 alpha,17 beta- and 16 beta,17 alpha-glycols approximately in the equal ratio. The delta 5-epoxidation of delta 16-ANDO by microsomes occurred only under the conditions that lipid peroxidation took place. Direct evidence was obtained for the participation of microsomal lipid hydroperoxides in the epoxidation of delta 16-ANDO by using photochemically prepared hydroperoxides of phospholipids separated from the hepatic microsomes. The hydroperoxides generated active oxygens, tentatively assigned as alk(ylper)oxy radicals, by the action of ferrous ion and epoxidized delta 16-ANDO to afford the 5,6- and 16,17-epoxides. The Fe2+-ADP-mediated epoxidation of delta 16-ANDO by the phospholipid hydroperoxides occurred preferentially at delta 5 to delta 16 and afforded the 5,6 beta-epoxide in a higher ratio than the 5,6 alpha-epoxide, similar to the Fe2+-ADP-mediated microsomal epoxidation, while the alpha-epoxide was preferentially formed to the beta-epoxide for delta 16 in the epoxidation by both systems.